This grant supports our studies of yeast RNA polymerase II (pol II) transcription. The long range goal of the work has been and remains to discover the mechanism of transcription and its regulation. The chief obstacle to progress has long been the requirement for abundant, monodisperse, pure GTFs and Mediator for mechanistic studies. Our work over the past twenty years has removed this obstacle, with the most intractable components, TFIIH and Mediator, having been obtained in suitable form during the previous project period. Three advances form the basis for the present proposal: our isolation in stable, homogeneous, fully functional form of a complete PIC (giant complex of pol II, all GTFs, and promoter DNA); our success, after decades of failed attempts, in establishing a gene activation system, starting from a gene isolated as chromatin in the repressed state, and giving rise to a high level of transcription; and developments both in mass spectrometry, giving 100-fold greater sensitivity, and in electron microscopy, stemming from our first synthesis of molecularly defined gold nanoparticles. Specific aims for the next project period are three-fold: to fractionate our gene activation system to elucidate the remodeling of chromatin structure for transcription, with the use of electron microscopy and mass spectrometry; and to investigate transcription initiation in a complete PIC by walking the early transcript and by single molecule analysis.